Methods for rational pegylation of proteins

Abstract

The present invention relates to the use of simulation technology to rationally optimize the locations and sizes of attached polymeric moieties for modification of therapeutic proteins and the proteins generated from this method.

1. A method of identifying at least one favorable attachment site for a polymeric moiety on a target protein comprising:
 a) inputting a set of coordinates into a computer for said target protein; 
 b) inputting a set of coordinates for said polymeric moiety; 
 c) selecting a criteria for said favorable attachment site based upon at least one desired characteristic, 
 d) using a simulation module comprising the steps of:
 i) computationally attaching conformers of said polymeric moiety to a plurality of amino acids in said target protein; and 
 ii) disallowing conformers at each of said amino acids on the basis of a distance cutoff; 
 
 e) selecting based upon said criteria one of said amino acids for attachment of said polymeric moiety; and 
 f) physically making and screening for said at least one desired characteristic at least one target protein with a polymeric moiety attached at said favorable attachment site amino acid position. 
 
   
   
     2. The method according to  claim 1 , wherein said method further comprises identifying a set of polymer sizes for attachment to said favorable attachment site. 
   
   
     3. The method according to  claim 1 , wherein the amino acid at said position is a non-naturally occurring amino acid. 
   
   
     4. The method according to  claim 3 , wherein said non-naturally occurring amino acid is p-acetyl-L-phenylalanine. 
   
   
     5. The method according to  claim 1 , wherein said polymeric moiety for attachment is pharmaceutically acceptable. 
   
   
     6. The method according to  claim 5 , wherein said polymeric moiety for attachment is a polyethylene glycol (PEG). 
   
   
     7. The method according to  claim 6 , wherein said PEG is a PEG derivative. 
   
   
     8. The method according to  claim 1 , wherein said polymeric moiety for attachment has a range of about 1000 daltons to about 100,000 daltons. 
   
   
     9. The method according to  claim 1 , wherein said polymeric moiety for attachment is branched. 
   
   
     10. The method according to  claim 1 , wherein said polymeric moiety for attachment is unbranched. 
   
   
     11. The method according to  claim 1 , wherein said polymeric moiety for attachment is labile. 
   
   
     12. The method according to  claim 1 , wherein said protein is a therapeutic protein. 
   
   
     13. The method according to  claim 1 , wherein said protein is selected from the group consisting of a human erythropoeitin (EPO), a human tumor necrosis factor (TNF), a human growth hormone (hGH), a human interferon (IFN), a human granulocyte colony stimulating factor (G-CSF) and bone morphogenic protein-7 (BMP7). 
   
   
     14. The method according to  claim 1 , wherein said simulation module includes Monte Carlo (MC) simulations, Molecular Dynamics (MD) simulations or combinations thereof. 
   
   
     15. The method according to  claim 1 , further comprising substituting at least one amino acid at said favorable attachment site with a cysteine residue. 
   
   
     16. The method according to  claim 1 , further comprising substituting at least one lysine or histidine residue of said protein with any other amino acid residue.